Nitrogen fixation plays a vital role in agricultural production by making atmospheric nitrogen available in a form that can be used by plants. In plants of the Leguminoseae family, the symbiotic interaction between the plants and nitrogen-fixing bacteria of the Rhizobiaceae family (“rhizobia”) enhances plant growth and crop yield. The symbiotic interaction is initiated when a plant releases flavonoid compounds that stimulate rhizobial bacteria in the soil to produce “Nod-factors.” Nod-factors are signaling compounds that induce the early stages of nodulation in plant roots, which lead to the formation of root nodules containing the nitrogen-fixing rhizobial bacteria. Although this process occurs naturally over time in legumes, agricultural procedures have been developed to begin the process earlier. These procedures include providing nitrogen-fixing bacteria to seeds or soil and applying Nod factors directly to seeds or soil prior to or at planting.
Nod factors have recently been shown to also enhance the germination, growth and yield of legumes and non-legumes through processes other than nodulation (U.S. Pat. No. 6,979,664; Prithivaraj et al., Planta 216: 437-445, 2003). Although the effects of Nod factors on nodulation have been widely studied and reviewed, e.g., Ferguson and Mathesius, J. Plant Growth Regulation 22: 47-72, 2003, the mechanisms for Nod factor effects independent of nodulation are not well understood. Application of Nod factors to seeds of legumes and non-legumes stimulates germination, seedling emergence, plant growth and yield in crop and horticultural plant species, e.g., as described in U.S. Pat. No. 6,979,664 and U.S. Pat. No. 5,922,316. Nod factors have also been shown to enhance root development (Olah, et al., The Plant Journal 44:195-207, 2005). Foliar application of Nod factors has also been demonstrated to increase photosynthesis (U.S. Pat. No. 7,250,068), and fruiting and flowering (WO 04/093,542) in crop and horticultural plant species.
Nod factors are lipo-chitooligosaccharide compounds (LCD's). They consist of an oligomeric backbone of β-1,4-linked N-acetyl-D-glucosamine (“GlcNAc”) residues with an N-linked fatty acyl chain at the nonreducing end. LCO's differ in the number of GlcNAc residues in the backbone, in the length and degree of saturation of the fatty acyl chain, and in the substitutions of reducing and nonreducing sugar residues. LCO structure is characteristic for each rhizobial species, and each strain may produce multiple LCO's with different structures. LCD's are the primary determinants of host specificity in legume symbiosis (Diaz, Spaink, and Kijne, Mol. Plant-Microbe Interactions 13: 268-276, 2000).
LCO synthesis can be stimulated by adding the appropriate flavonoid, for a given genus and species of rhizobium during growth of the bacteria. The flavonoid molecules bind to the rhizobium and turn on bacterial genes for the production of specific LCD's which are released into the fermentation medium. In nature, leguminous plants release the appropriate flavonoid, which binds to soil rhizobia, turning on genes for LCO production. These LCO's are released by bacteria into the soil, bind to the roots of leguminous plants, and initiate a cascade of plant gene expression that stimulates formation of nitrogen-fixing nodule structures on legume roots. Alternatively, modified and synthetic LCO molecules can be produced through genetic engineering or chemical synthesis. Synthetic LCD's of the same molecular structure interact with plants and stimulate nodulation in the same manner as naturally produced molecules.
Chitins and chitosans, which are major components of the cell walls of fungi and the exoskeletons of insects and crustaceans, are also composed of GlcNAc residues. These compositions have been applied to seeds, roots, or foliage of a broad spectrum of crop and horticultural plants. Chitin and chitosan compositions enhance protection against plant pathogens, in part, by stimulating plants to produce chitinases, enzymes that degrade chitin (Collinge, et al., The Plant Journal 3: 31-40, 1993).
Flavonoids are phenolic compounds having the general structure of two aromatic rings connected by a three carbon bridge. Flavonoids are produced by plants and have many functions, e.g., as beneficial signaling molecules, and as protection against insects, animals, fungi and bacteria. Classes of flavonoids include chalcones, anthocyanidins, coumarins, flavones, flavanols, flavonols, flavanones, and isoflavones. (Jain and Nainawatee, J. Plant Biochem. & Biotechnol. 11: 1-10, 2002; Shaw, et al., Environmental Microbiol. 11: 1867-1880, 2006.)